Myoglobin && Hemoglobin Myoglobin, hemoglobin, cytochromes, catalase & tryptophan pyrrolase are hemoporteins. The heme present in myoglobin & hemoglobin is cyclic tetrapyrol compound contains iron in the ferrous state( Fe+2), this iron has six coordination positions, four of them bind to the four nitrogen atoms of pyrrole in a same plane while the fifth & sixth coordination positions are directed perpendicular to & directly above & below the plane of the heme ring. Oxidation of Fe+2 of myoglobin & hemoglobin to Fe+3 destroys their biological activity. Cyanide & carbon monoxide kill the human because they disrupt the physiologic function of the hemoprotein like cytochrome oxidase & hemoglobin. Biological function of hemoglobin & myoglobin Myoglobin store oxygen in the heart & skeletal muscles as a reserve against oxygen deprivation while hemoglobin transport O2 to the tissues & returns CO2 & protons to the lung. Myoglobin Myoglobin is a hemoprotein present in the heart & skeletal muscles that store oxygen as a reserve against oxygen deprivation. Its globin fraction is a monomer (monomeric protein) consists from 153- aminoacyl residue folds in eight α-helices conformation forming a compact shape of myoglobin ((globular protein)), each helix contains 7-20 aminoacyl residues, starting from amino terminal these helices are termed A-H. As any other globular protein the surface of myoglobin is polar with only two exceptions ((Histidines at E7 & F8 location)) while the interior contains only nonpolar residues. The heme of myoglobin lies in a crevice between E & F helices with a fifth coordination position of the iron is linked to nitrogen of proximal histidine((His F8)) while the distal histidine ((His E7)) lies on the side of the heme ring opposite to His F8. The sixth coordination position of iron is linked to oxygen in oxygenated myoglobin. The iron of unoxygenated myoglobin lies 0.03 nm ((0.3 Ao)) outside the plane of heme toward proximal histidine ((His F8)) while in oxygenated 1 myoglobin when O2 occupies the sixth coordination position, the iron move to within 0.01 nm ((0.1 Ao)) of the plane of heme. 2 The presence of distal histidine ((His E7)) is important in oxygenation of myoglobin as follow: When O2 binds to myoglobin, the bond between the first oxygen atom & Fe++ is perpendicular to the plane of heme ring while the bond linking the first & second oxygen atoms lies at an angle of 1210 to the plane of the heme due to presence of distal histidine creating what is called hindered environment. In spite of isolated heme binds carbon monoxide {CO} 25 000 times more strongly than O2, the presence of hindered environment restrict the binding of all atoms of CO to the sixth coordination position in a perpendicular direction to the plane of heme as preferred by CO, therefore, reduce the strength of heme-CO bond to about 200 times that of the heme-O2 bond at which level the great excess of O2 over CO made only about 1 % of myoglobin combined with CO. Note:-The explanation of hindered environment is applied also to hemoglobin binding with O2. Oxygen Dissociation (Binding) Curves It represents the relationship between partial pressure of oxygen (Po2) & % of oxygen saturation. Po2 values: -In arterial blood is about 100 mmHg. -In mixed venous blood is about 40 mmHg. -In capillary (active muscle) is about 20 mmHg. -Minimum Po2 required for cytochrome oxidase (important in the respiratory chain action) activity is about 5 mmHg. The oxygen dissociation curve of myoglobin is hyperbolic shape, this explain why myoglobin act as storage & not as transporter of oxygen because even at low Po2 (20 mmHg in active muscle), only small fraction of oxygen is lost by myoglobin, however, when exercise lower Po2 of 3 muscle tissues to 5 mmHg which is a minimum Po2 required for cytochrome oxidase, the myoglobin start to release oxygen. The oxygen dissociation curve of hemoglobin is sigmoid shape, this explain why Hb start to release oxygen at a higher level of Po2 value than that of myoglobin, therefore, it acts as a transporter of oxygen. Hemoglobin (Hb) Hemoglobins are hemoproteins present in RBC, their globin fraction is a tetramer consist of pair of two different polypeptide subunits as follow: -HbA is α2 β2 :represents more than 95% of total Hb in healthy adult. -HbA2 is α2 δ2:represents up to 4% of total Hb in healthy adult. -HbF is α2 γ2 :represents up to 2% of total Hb in healthy adult. -HbS is α2 S2: present in sickle cell anemia. Similarities are present between HbA subunits & myoglobin in the location of heme & the presence of amino acids with similar properties at comparable locations; however, there are some differences in the number & kinds of amino acids & the number of helical regions. There is one heme bind to each of four Hb-subunits, therefore, there are four heme molecules present in one molecule of Hb making Hb can binds four molecules of oxygen ((one/heme)), this binding is cooperative which mean that once first oxygen molecule binds to Hb-tetramers the binding of second oxygen molecule is more readily & so on. P50 of Hb Define as a value of Po2 at which half of a given Hb is saturated with oxygen ((50% oxygen saturation)). Values of P50 differ according to the type of Hb e.g: -HbA P50 = 26 mmHg -HbF P50= 20 mmHg 4 This mean HbF has a higher affinity to oxygen than HbA, therefore, HbF which present in the embryonic life can extract oxygen from HbA in the mother's blood, however, because of its higher oxygen affinity ((give less oxygen to tissues)) it replaced gradually by HbA from third trimester until several weeks postpartum in which replaced by HbA that gives more oxygen to tissues. Oxygenation of Hemoglobin The binding of the first O2 molecule to deoxyHb (unoxygenated Hb) lead to series of changes causing cooperative property of Hb. These changes occur through the following steps: Step ((1)).. Movement of proximal histidine ((F8)) & iron toward the plane of the heme. Step((2))..Rupture of salt bridges between the carboxyl terminal residues of all four subunits. 5 Step((3))..One pair of α/β subunits rotates 150 with respect to other pair. Step((4))..The previous three steps cause transition of Hb from the low- affinity T (taut) state to the high-affinity R (relaxed) state which binds easy to oxygen. Transport of CO2 & protons CO2 & protons are transported from peripheral tissues to the lung as follow: A))-Transport of CO2: Hb carries CO2 as carbamates( formed with the amino terminal nitrogens of the polypeptide chains( so carbamates change the charge of amino terminals of Hb from positive to negative favoring salt-bond formation between the α & β chains which stabilizes the T-state. This process accounts for about 15% of the CO2 transport in the venous blood while the remaining of CO2 is carried as bicarbonate through the formation of carbonic acid. 6 B))-Transport of protons: Deoxyhemoglobin binds one proton for every two O2 molecules released share in buffering of blood, this process with carbamation was further stabilizes the T-state so enhance the delivery of oxygen to the tissues while in the lung the process is reversed, this reciprocal coupling of proton & oxygen binding is termed the “Bohr effect” which depend on cooperative interaction between the hemes of the Hb tetramer which is absent in myoglobin because its monomer, therefore, myoglobin exhibit no Bohr effect. Adaptation to low Po2 The low Po2 in the peripheral tissues can occur for example in the long term exposure to high altitude & in the smokers; the main human adaptations to this condition are the following: 1- A low Po2 in the peripheral tissues promotes the synthesis of 2,3- bisphosphoglycerate (BPG) in the erythrocytes from the glycolytic intermediate1,3-bisphosphoglycerate. One molecule BPG binds one molecule of HbA mainly at β-chain forming additional salt-bridges that further stabilized the deoxygenated ((T state)) of HbA so reducing the HbA affinity to oxygen, since HbF is devoid of β-chain, therefore, it cannot form salt-bridges with BPG making its affinity to oxygen higher than HbA. 7 2- Increase production of erythropoietin by the kidney which stimulates bone marrow to produce RBC & Hb. Hemoglobinopathy When mutation of human hemoglobin affects its biological function the condition is known as “Hemoglobinopathy”. Examples are: 1- Methemoglobin & Hemoglobin M. 2- Hemoglobin S ((HbS)). 3- Thalassemias. Methemoglobin & Hemoglobin M In Methemoglobin, the heme iron is in the ferric state so neither bind nor transport oxygen, it arise either from: 1-Side effect of drugs such as sulphonamide which cause oxidation of ferrous to ferric state. 2-Low activity of methemoglobin reductase enzyme which reduce ferric to ferrous state. 3-Hemoglobin M (HbM); In which His F8 has been replaced by tyrosine, therefore, iron of HbM forms a tight ionic complex with tyrosine that stabilizes the ferric form. Hemoglobin S ((HbS)) In this hereditary condition, the S-subunit of HbS (α2 S2) is formed when the nonpolar amino acid ((valine)) has replaced the polar surface amino acid ((glutamic acid)) at position 6 of the β subunit of the HbA generating a hydrophobic ((sticky patch)). The sticky patch is found in the S-subunit 8 of both oxyHbS & deoxyHbS lead to a pathological condition known as “sickle cell anemia”. The proportions of HbA that is converted into HbS are varied, the more HbA is converted into HbS the more sever the condition is, because although both deoxy HbA & HbS have a complementary sticky patch, however, at low Po2 as in high altitudes the deoxy HbS that contains both sticky & complementary sticky patches can polymerize to form long insoluble fibers while binding to deoxyHbA terminates fiber polymerization because HbA lacks sticky patch necessary to bind another Hb molecule.